The present invention relates to a method for enhancing the level of perfusion of blood to a target tissue, a method for treating a target tissue suffering from or at risk of suffering from ischemic damage, and a method of inducing angiogenesis in a target tissue.
Angiogenesis, the growth of new blood vessels, is a complex process involving the disruption of vascular basement membranes, migration and proliferation of endothelial cells, and subsequent blood vessel formation and maturation. Several mediators are known to elicit angiogenic responses, and administration of these mediators promotes revascularization of ischemic tissues. Vascular endothelial growth factor (VEGF protein) is one of the most specific of the known angiogenic mediators due to localization of its receptors almost exclusively on endothelial cells. Receptors for VEGF are upregulated under ischemic conditions, and the administration of recombinant VEGF augments the development of collateral vessels and improves function in peripheral and myocardial ischemic tissue.
However, delivery of VEGF protein remains a significant challenge. The half-life of VEGF protein is very short; the administration of high doses of VEGF protein is associated with hypotension, and systemic administration of VEGF protein can cause promiscuous induction of angiogenesis in tissues other than that which has been targeted. Promiscuous induction of angiogenesis can cause blindness, increase the aggressiveness of tumor cells, and lead to a multitude of other negative side-effects. Furthermore, the quantity of VEGF protein delivered is important. If too little VEGF protein is delivered, angiogenesis will not be induced, and a significant therapeutic benefit will not be achieved. If too much VEGF protein is delivered, the formation of disorganized vasculature beds, loss of function in the affected tissue, and promiscuous angiogenesis can result.
Additionally, induction of angiogenesis via administration of liposomes and/or xe2x80x9cnakedxe2x80x9d DNA comprising a DNA encoding an angiogenic peptide also suffer from numerous disadvantages. Specifically, both liposomal and xe2x80x9cnakedxe2x80x9d DNA forms of delivery are less efficient than viruses at transferring genes to cells, are inefficient at integrating genes into the host genome, and are difficult to target to specific tissues.
In view of the foregoing, there exists a need for an effective method of inducing angiogenesis in a target tissue. The present invention provides such a method. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The present invention provides a method for enhancing the level of perfusion of blood to a target tissue comprising administering, via multiple applications to the target tissue, a dose of a pharmaceutical composition comprising (a) a pharmaceutically acceptable carrier and (b) an adenoviral vector comprising a DNA encoding an angiogenic peptide, such that the level of perfusion of blood to the target tissue is enhanced. Also provided is a method for treating a target tissue suffering from or at risk of suffering from ischemic damage comprising administering, via multiple applications to the target tissue, a dose of a pharmaceutical composition comprising (a) a pharmaceutically acceptable carrier and (b) an adenoviral vector comprising a DNA encoding an angiogenic peptide, such that the dose has a therapeutic or prophylactic effect on the target tissue. Further provided is a method for inducing angiogenesis in a target tissue comprising administering, via multiple applications to the target tissue, a dose of a pharmaceutical composition comprising (a) a pharmaceutically acceptable carrier and (b) an adenoviral vector comprising a DNA encoding an angiogenic peptide, such that angiogenesis is induced in the target tissue. Additionally provided is a method for inducing collateral blood vessel formation in a target tissue affected by or at risk of being affected by a vascular occlusion comprising administering to the target tissue a dose of a pharmaceutical composition comprising (a) a pharmaceutically acceptable carrier and (b) an adenoviral vector comprising a DNA encoding an angiogenic peptide, such that the adenoviral vector contacts a region including the source, the terminus, and an area therebetween for the collateral blood vessel formation, and collateral blood vessel formation is induced.
The invention may best be understood with reference to the following detailed description of the preferred embodiments. The present invention provides a method for enhancing the level of perfusion of blood to a target tissue, a method for treating a target tissue suffering from or at risk of suffering from ischemic damage, a method for inducing angiogenesis in a target tissue, and/or a method for inducing collateral blood vessel formation in a target tissue affected by or at risk of being affected by a vascular occlusion. Each of these methods involves administering, via multiple applications to the target tissue, a dose of a pharmaceutical composition comprising (a) a pharmaceutically acceptable carrier and (b) an adenoviral vector comprising a DNA encoding an angiogenic peptide, such that the level of perfusion of blood to the target tissue is enhanced, the dose has a therapeutic or prophylactic effect on the target tissue, angiogenesis is induced in the target tissue, and/or the adenoviral vector contacts a region including the source, the terminus, and an area therebetween for collateral blood vessel formation, and collateral blood vessel formation is induced.
Induction of Angiogenesis
By the term xe2x80x9cinducing angiogenesis,xe2x80x9d it is meant that angiogenesis is either initiated or enhanced. Therefore, for example, when the target tissue is not already undergoing angiogenesis, the present method provides for the initiation of angiogenesis in the target tissue. However, when the target tissue is already undergoing angiogenesis, the present method provides a means by which the level of angiogenesis is enhanced or heightened.
Target Tissue
Any suitable tissue can be subject to administration within the context of the present invention. Preferably, the target tissue comprises receptors capable of binding the angiogenic peptide encoded by the DNA; more preferably, the target tissue comprises VEGF receptors. Most preferably, the target tissue comprises endothelial cells. Generally, the target tissue will be a part of or form a discrete organ, e.g., a muscle, such as the heart.
Typically, the target tissue will be suffering from or be at risk of suffering from ischemic damage which results when the tissue is deprived of an adequate supply of oxygenated blood. The interruption of the supply of oxygenated blood is often caused by a vascular occlusion. Such vascular occlusion can be caused by arteriosclerosis, trauma, surgical procedures, disease, and/or other indications. There are many ways to determine if a tissue is at risk of suffering ischemic damage from undesirable vascular occlusion. Such methods are well known to physicians who treat such conditions. For example, in myocardial disease these methods include a variety of imaging techniques (e.g., radiotracer methodologies such as 99mTc-sestamibi scanning, x-ray, and MRI scanning) and physiological tests. Therefore, induction of angiogenesis in tissue affected by or at risk of being affected by a vascular occlusion is an effective means of preventing and/or attenuating ischemia in such tissue. As a result, although any suitable tissue can be targeted for the induction of angiogenesis, the target tissue is preferably one which is affected by or at risk of being affected by a vascular occlusion.
For example, the blood supply to discrete organs such as the brain, heart, pancreas, entire limbs, or generalized areas of the body, such as a foot, can be attenuated by disease, trauma, surgery, or other events. The alleviation of such attenuated blood supply regardless of its origin is contemplated by the present invention. Thus, prevention or alleviation of damage from indications such as myocardial ischemia and stroke are fully contemplated. Additionally, the planning of a surgical procedure can be predictive of the interruption of blood supply through a particular portion of a patient""s vasculature. Prior treatment according to the present method can substantially improve the desired outcome of these surgeries. In that case, treatment preferably occurs about one day to about six weeks before said surgery, and more preferably about two to about fourteen days prior to surgery.
Administration of Angiogenic Vector
As previously stated, the induction of angiogenesis via the systemic administration of angiogenic peptides, such as VEGF protein, can lead to promiscuous induction of angiogenesis which, for example, can cause blindness and increase the aggressiveness of tumor cells. Therefore, in order to attenuate or prevent such negative side-effects it is desirable to induce angiogenesis only in the tissue which requires it (i.e., the target tissue).
The present invention involves the administration of an adenoviral vector comprising a DNA encoding an angiogenic peptide in a localized manner to the target tissue. While any suitable means of administering the angiogenic vector to the target tissue can be used within the context of the present invention, preferably, such a localized administration to the target tissue is accomplished by directly injecting the angiogenic vector into the target tissue or by topically applying the angiogenic vector to the target tissue. By the term xe2x80x9cinjecting,xe2x80x9d it is meant that the angiogenic vector is forcefully introduced into the target tissue. Any suitable injection device can be used within the context of the present invention. Such injection devices include, but are not limited to, that described in U.S. Pat. No. 5,846,225, which is directed to a gene transfer delivery device capable of delivering simultaneous multiple injections. Another example of an injection device which can be used within the context of the present invention includes minimally invasive injection devices. Such devices are capable of accessing the heart, for example, through small incisions of less than 5 inches and are designed to provide injections through a single lumen, in contrast to the multiple injection device described above. To allow for the need for multiple injections with a specific geometry, a marking system can be employed so that the sites of previous injections are well delineated. Minimally invasive injection devices can comprise injector tips which are flexible and steerable to allow access via small incisions to the curved outer surface of the heart, for example, which exists at varying angles with respect to the limited aperture window required with minimally invasive surgeries.
Furthermore, the angiogenic vector can be administered to any suitable surface, either internal or external, of the target tissue. For example, with respect to directly injecting the angiogenic vector into cardiac tissue, it is contemplated that such an injection can be administered from any suitable surface of the heart (i.e., endocardially and/or epicardially). However, it is desirable that whatever means of administering the angiogenic vector is chosen, the induction of angiogenesis in non-targeted tissue is minimized.
While administration of a dose of the angiogenic vector can be accomplished through a single application (e.g., a single injection or a single topical application) to the target tissue, preferably, administration of the dose is via multiple applications of the angiogenic vector. The multiple applications can be 2, 3, 4, 5, or more applications, preferably 5 or more applications, more preferably 8 or more applications, and most preferably at least 10 (e.g., 10, 15, or 20) applications. Multiple applications provide an advantage over single applications in that they can be manipulated by such parameters as a specific geometry defined by the location on the target tissue where each application is administered. The administration of a single dose of the angiogenic vector via multiple applications can be better controlled, and the effectiveness with which any given dose is administered can be maximized. In this way, too, the undesirable effects associated with administration of a single point application of a large dose can be minimized.
The specific geometry of the multiple applications is defined by the location on the target tissue, either in two- or three-dimensional space, where each application of the angiogenic vector is administered. The multiple applications preferably are spaced such that the points of application are separated by up to about 4 cm (e.g., about 0.5-4 cm), more preferably up to about 3 cm (e.g., about 1-3 cm), and most preferably up to about 2 cm (e.g., about 1-2 cm). With respect to the specific geometry of the multiple applications in two-dimensional space, the specific geometry is defined by a plane (i.e., a cross-section of the target tissue) in which the multiple applications lie. The plane defined by the multiple applications can lie at a constant distance from the surface of the target tissue (i.e., substantially parallel to the surface of the target tissue), the depth of the plane, or, alternatively, the plane can lie at an angle with respect to the surface of the target tissue. Preferably, a single application will be administered for about every 0.5-15 cm2 of the plane, more preferably for about every 1-12 cm2 of the plane, and most preferably for about every 1.5-7 cm of the plane. The depth of the plane is preferably about 1-10 mm, more preferably about 2-7 mm, and most preferably about 3-5 mm. In three-dimensional space, a single application preferably is administered for up to about 50 cm3 (e.g., about 0.5-50 cm3) of target tissue, more preferably for up to about 35 cm3 (e.g., about 1-35 cm3) of target tissue, and most preferably for up to about 15 cm3 (e.g., about 3-15 cm3) of target tissue. Furthermore, the multiple applications can define any suitable pattern or specific geometry. Therefore, for example, in two-dimensional space, the multiple applications can define a square whereas in three-dimensional space the multiple applications can define a cube.
Another parameter of the multiple applications which can be manipulated is the time differential between each application. Preferably, each of the multiple applications is administered within about 10 minutes (e.g., about 0.5-10 minutes) of each other, more preferably within about 8 minutes (e.g., about 0.5-8 minutes) of each other, and even more preferably within about 6 minutes (e.g., about 1-6 minutes) of each other. Most preferably, all of the multiple applications of the single dose are administered within the aforesaid time frames. Optimally, each of the multiple applications is administered substantially simultaneously.
By manipulating both the specific geometry and the time differentials of the multiple applications, the induction of angiogenesis in non-targeted tissue can be minimized.
When administering the angiogenic vector to a target tissue which is affected by or at risk of being affected by a vascular occlusion, it is desirable that the administration is such that the angiogenic vector is able to contact a region reasonably adjacent to the source and the terminus for the collateral blood vessel formation, as well as the area therebetween, which will function as a bypass to the vascular occlusion. It is not believed to be necessary to have the angiogenic vector actually contact the precise sites of the source and the terminus for the collateral blood vessel formation. However, within the context of multiple applications of the angiogenic vector, it is desirable that the specific geometry of the multiple applications be defined to allow the angiogenic vector to contact or reach a region including the source, the terminus, and the area therebetween for the collateral blood vessel formation, preferably to actually contact the precise sites of the source and the terminus for the collateral blood vessel formation, along with the area therebetween.
Furthermore, administration of the angiogenic vector to the target tissue can be accomplished either in vivo or ex vivo. Therefore, for example, the target tissue can be removed from the recipient of the present inventive method, can be treated with the angiogenic substance, and then can be reimplanted into the recipient. Ex vivo administration of the angiogenic substance to the target tissue also helps to minimize undesirable induction of angiogenesis in non-targeted tissue.
Angiogenic Vector
As previously stated, the delivery of VEGF protein as an angiogenic substance to tissue remains a significant challenge due, in large part, to its very short half-life. However, by utilizing an adenoviral vector comprising a DNA encoding an angiogenic peptide as the angiogenic substance, it is possible to infect host cells and thereby induce the sustained, predictable, and effective production of an angiogenic peptide for about a week. After about a week, the adenoviral vector ceases to produce the angiogenic peptide and, to that extent, the present invention provides a self-terminating method of inducing angiogenesis.
Adenoviral vectors are preferred because, unlike plasmids and other viral vectors (e.g., herpes simplex virus), adenoviral vectors achieve gene transfer in both dividing and nondividing cells, with high levels of protein expression in cardiovascular relevant sites such as myocardium, vascular endothelium, and skeletal muscle. Furthermore, the gene transferred by an adenoviral vector functions in an epi-chromosomal position and thus carries little risk of inappropriately inserting the transferred gene into a critical site of the host genome. The adenoviral vector also is preferably deficient in at least one gene function required for viral replication. Preferably, the adenoviral vector is deficient in at least one essential gene function of the E1 region of the adenoviral genome, particularly the E1a region, more preferably, the vector is deficient in at least one essential gene function of the E1 region and part of the E3 region (e.g., an XbaI deletion of the E3 region) or, alternatively, the vector is deficient in at least one essential gene function of the E1 region and at least one essential gene function of the E4 region. However, adenoviral vectors deficient in at least one essential gene function of the E2a region and adenoviral vectors deficient in all of the E3 region also are contemplated here and are well known in the art. Adenoviral vectors deleted of the entire E4 region can elicit lower host immune responses. Suitable replication deficient adenoviral vectors are disclosed in U.S. Pat. No. 5,851,806 and PCT International Publication No. WO 95/34671. For example, suitable replication deficient adenoviral vectors include those with a partial deletion of the E1a region, a partial deletion of the E1b region, a partial deletion of the E2a region, and a partial deletion of the E3 region. Alternatively, the replication deficient adenoviral vector can have a deletion of the E1 region, a partial deletion of the E3 region, and a partial deletion of the E4 region.
Furthermore, the viral vector""s coat protein can be modified so as to incorporate a specific protein binding sequence, as described in U.S. Pat. No. 5,432,075, or the viral vector""s coat protein can be modified so as to decrease the viral vector""s ability or inability to be recognized by a neutralizing antibody directed against the wild-type coat protein, as described in PCT International Publication No. WO 98/40509.
Any DNA encoding an angiogenic peptide and operably linked to suitable expression signals can be used within the context of the present invention. Whereas the DNA can be operably linked to any suitable set of expression signals, preferably, the expression of the DNA is under the control of the cytomegalovirus (CMV) immediate early promoter.
Additionally, the DNA can encode any suitable angiogenic peptide. Preferably, the angiogenic peptide is a VEGF protein, and more preferably, the angiogenic peptide is VEGF121, VEGF145, VEGF165, VEGF189 or a mammalian counterpart, which are variously described in U.S. Pat. No. 5,332,671 (Ferrara et al.), U.S. Pat. No. 5,240,848 (Keck et al.), and U.S. Pat. No. 5,219,739 (Tischer et al.). Most preferably, because of their higher biological activity, the angiogenic peptide is VEGF121 or VEGF165, particularly VEGF121. A notable difference between VEGF121 and VEGF165 is that VEGF121 does not bind to heparin with a high degree of affinity as does VEGF165. Generally, VEGF moieties are advantageous over other angiogenic peptides because VEGF proteins do not induce the growth of tissues not involved in the production of new vasculature. Other angiogenic peptides include VEGF II, VEGF-C, FGF-4, angiogenin, angiogenin-2, and P1GF, which are variously described in U.S. Pat. No. 5,338,840 (Bayne et al.) and U.S. Pat. No. 5,532,343 (Bayne et al.), International Patent Application WO 95/24473 (Hu et al.), European Patent Documents 476 983 (Bayne et al.), 506 477 (Bayne et al.), and 550 296 (Sudo et al.), and Japanese Patent Documents 1038100, 2117698, 2279698, and 3178996.
The adenoviral vector also can include a DNA encoding an angiogenic peptide receptor. Suitable angiogenic peptide receptors include, for example, FLT-1, FLK-1, and FLT-4. Indeed, in certain embodiments, the adenoviral vector can utilize a DNA encoding an angiogenic peptide receptor in place of, rather than in addition to, the DNA encoding an angiogenic peptide.
The DNA, operably linked to expression signals and encoding the angiogenic peptide, can be inserted into any suitable region of the adenoviral vector as an expression cassette. In that respect, the skilled artisan will readily appreciate that there are certain advantages to using an adenoviral vector deficient in some essential gene region of the adenoviral genome inasmuch as such a deficiency will provide room in the vector for a transgene and will prevent the virus from replicating. Preferably, the DNA segment is inserted into the E1 region of the adenoviral vector. Whereas the DNA segment can be inserted as an expression cassette in any suitable orientation in any suitable region of the adenoviral vector, preferably, the orientation of the DNA segment is from right to left. By the expression cassette having an orientation from right to left, it is meant that the direction of transcription of the expression cassette is opposite that of the region of the adenoviral vector into which the expression cassette is inserted.
An adenoviral vector illustrative of the present inventive vector is deficient in the E1a region, part of the E1b region, and part of the E3 region of the adenoviral genome and contains the DNA encoding human VEGF121 or human VEGF165 under the control of the CMV immediate early promoter in the E1 region of the adenoviral genome. Such a vector supports in vivo expression of VEGF that is maximized at one day following administration and is not detectable above baseline levels as little as one week after administration. This is ideal inasmuch as it is sufficient to provide substantial growth of new vasculature while minimizing adverse neovascularization at distal sites. In that regard, when this vector is locally administered to a target tissue, no detectable VEGF expression can be detected in blood serum using standard ELISA monitoring assays.
Advantageously, local administration to a target tissue of adenoviral vectors encoding human VEGF121 or VEGF165 in the E1 region of the adenoviral genome are able to increase blood flow at least 3-fold in the extremities of mammals (e.g., the hindlimb of Sprague-Dawley rats) with iliac and femoral artery ligations.
Pharmaceutical Composition
The angiogenic vector desirably is administered to the target tissue in a pharmaceutical composition which comprises a pharmaceutically acceptable carrier and the angiogenic vector.
Any suitable pharmaceutically acceptable carrier can be used within the context of the present invention, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition is to be administered and the particular method used to administer the composition. Formulations suitable for injection include aqueous and non-aqueous solutions, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Preferably, the pharmaceutically acceptable carrier is a buffered saline solution.
Although any suitable volume of carrier can be utilized within the context of the present invention, preferably, the angiogenic vector is administered in small volumes of carrier so that the tissue to be vascularized (i.e., the target tissue) is perfused with the angiogenic vector but the angiogenic vector is not carried by the blood, lymphatic drainage, or physical mechanisms (e.g., gravitational flow or osmotic flow) to tissues which have not been targeted.
In the case of most applications, particularly to discrete organs such as with respect to human myocardial injections, the volume administered is preferably less than 20 ml (e.g., about 0.1-20 ml) per each administration and more preferably less than about 2.5 ml (e.g., about 0.5-2.5 ml) per each administration.
Dosage
The determination of the proper dosage of the angiogenic vector can be easily made by those of ordinary skill in the art. However, generally, certain factors will impact the dosage which is administered.
Although the proper dosage is such that angiogenesis is induced in the target tissue, preferably, the dosage is sufficient to have a therapeutic and/or prophylactic effect on target tissue which is affected by or at risk of being affected by a vascular occlusion which may lead to ischemic damage of the tissue. Additionally, the dosage should be such that induction of angiogenesis in non-targeted tissue is minimized.
The dosage also will vary depending upon the angiogenic substance to be administered. Specifically, the dosage will vary depending upon the particular vector and DNA, encoding and controlling the expression of the angiogenic peptide in the vector, which are utilized. A dose typically will be at least about 1xc3x97106 pfu (e.g., 1xc3x97106-1xc3x971013 pfu) to the target tissue, e.g., a discrete organ, such as a human heart. The dose preferably is at least about 1xc3x97107 pfu (e.g., about 1xc3x97107-1xc3x971013 pfu), more preferably at least about 1xc3x97108 pfu (e.g., about 1xc3x97108-1xc3x971011 pfu), and most preferably at least about 1xc3x97109 pfu (e.g., about 1xc3x97109-1xc3x971010 pfu). The dose typically is for a volume of targeted tissue of about 100 cm3, more typically about 150 cm3. The dose is administered via multiple applications, and, as such, is divided among the multiple applications. Thus, if the dose is administered via 10 administrations, each administration involves about 1xc3x97105-1xc3x971012 pfu. Preferably, each application involves about 1xc3x97106-1xc3x971012 pfu, more preferably about 1xc3x97107-1xc3x971010 pfu, and most preferably about 1xc3x97108-1xc3x97109 pfu. For purposes of considering the dose in terms of particle units (pu), also referred to as viral particles, it can be assumed that there are 100 particles/pfu (e.g., 1xc3x971012 pfu is equivalent to 1xc3x971014 pu). In a single round of vector administration, using, for example, an adenoviral vector deleted of the E1a region, part of the E1b region, and part of the E3 region of the adenoviral genome, wherein the vector carries human VEGF121 or VEGF165 under the control of a standard CMV immediate early promoter, about 107-1013 pfu, preferably about 109-1011 pfu, are administered to a targeted tissue (e.g., to a discrete organ containing the targeted tissue) with an estimated volume of about 150 cm3. Under these conditions, a substantial level of VEGF production is achieved in the target tissue without producing detectable levels of VEGF production in distal tissues.
Furthermore, with respect to multiple applications of the angiogenic vector, each application can be such that a dosage gradient is administered across the region defined by the multiple applications. Alternatively, each of the multiple applications can be such that a substantially uniform dose is administered across the region defined by the multiple applications.